Expression vectors, including expression plasmids are used commercially to produce gene products more efficiently than previously available methods such as extraction from natural sources and organic synthesis. Escherichia coli is widely used as a host in which such gene products may be produced using expression plasmids. A comprehensive discussion of expression vectors appears in P. H. Pouwels, B. E. Enger-Valk and W. J. Brammer, Cloning Vectors, Elsevier, publs (1985).
A number of control mechanisms which regulate the level of gene expression in prokaryotic vectors have been described. The regulatory process may operate at the transcriptional or translational level. At the transcriptional level control of gene expression is associated with inhibition or stimulation of mRNA synthesis.
One method of increasing the level of expression of a desired gene product is to use a vector in which the gene to be expressed is under the control of a strong promoter, and this is the predominant approach used in the biotechnology industry to express gene products at high levels.
The transcriptional control system consists of a DNA sequence contained in or adjacent to, a promoter which is capable of binding a repressor molecule. When the repressor molecule binds to a specific strain of DNA, transcription of DNA to mRNA is prevented. To initiate transcription of DNA to mRNA the repressor molecule has to be removed or dissociated from its binding site, and this is achieved by a specific chemical or physical stimulus. In many cases of transcriptional control an additional component is needed to drive transcription to its highest capacity. One example of such a component is cyclic AMP-receptor protein (CRP) which is involved in the regulation of many genes in catabolic pathways.
Expression plasmids containing the regulatable promoters .lambda. P.sub.L, trp or lac, have been extensively used in the art. Other promoters which have been used include the regulatable rec A and .lambda. P.sub.R promoters, the constitutive amp and lpp promoters, and artificial promoters created by fusion of two different DNA sequences or by chemical synthesis of DNA sequences.
The Escherichia coli deo operon has been studied by Hammer-Jesperson and co-workers. [K. Hammer-Jesperson and A. Munch-Peterson, Molec. gen. Genet. 137: 327-335 (1975); R. S. Buxton, H. Albrechtsen and K. Hammer-Jesperson, J. Mol. Biol. 114: 287-300 (1977); R. S. Buxton, K. Hammer-Jesperson and T. D. Hansen, J. Bacteriol. 136: 668-681 (1978); P. Valentin-Hansen and K. Hammer-Jesperson, J. Mol. Biol. 133: 1-17 (1979); and P. Valentin-Hansen, H. Aiba and D. Schumperli, The EMBO Journal 1: 317-322 (1982)].
As illustrated in FIG. 20, the deo operon consists of four closely-related genes encoding enzymes involved in nucleotide and deoxynucleotide catabolism, namely, deoxyriboaldolase (deo C), thymidine phosphorylase (deo A), phosphodeoxyribomutase (deo B) and purine nucleotide phosphorylase (deo D). Transcription of all four closely-related deo genes is regulated at two promoter/operator regions, deo P1 and deo P2 (or P1 and P2). When the transcription of a gene is controlled by the deo P1 and deo P2 promoters functioning in tandem, the term deo P1-P2 promoter or deo promoter/operator region is used.
Initiation of transcription from P1 is negatively controlled by the deo R repressor and is activated by the inducer deoxyribose-5-phosphate. Initiation of transcription from P2 is negatively controlled by the cyt R repressor, the inducer being cytidine. Activation of P2 depends on the cyclic AMP/cyclic AMP receptor protein complex, cAMP/CRP, although P2 may also be controlled by the deo R repressor. The two distal genes B and C in the deo operon are subject to an additional transcriptional control: an internal promoter/operator region P3, regulated by unknown control proteins and responding to induction by inosine and guanosine. The sequence of the deo P1-P2 promoter/operator region is known, and the entire deo P1-P2 promoter/operator region is approximately 760 bp long, with the two promoters being separated by a distance of about 600 bp. (P. Valentin-Hansen et al., The EMBO Journal 1: 317-322 (1982)).
Expression of a gene product under deo P2-driven transcription is very low in the presence of glucose and very high in the presence of other energy producing sources. Transcription driven by deo P1 is not subject to the glucose effect (catabolite repression), and also deo P1 is a much weaker promoter than deo P2. Therefore, one may manipulate expression of a gene product driven by the deo P1 promoter both by external means and by choosing Escherichia coli cells which possess no functional deo R repressor.
The entire deo operon has been cloned from .lambda. bacteriophage harboring the deo operon and the deo enzymes have been expressed in Escherichia coli host cells [M. Fischer and A. Short, Gene 17: 291-298 (1982)]. It was found that the enzyme activities of bacteria containing the cloned deo operon were amplified 500-50,000 fold over the level found in wild-type cells. The degree of amplification depends on host genotype. In host cells possessing both the deo R and cyt R repressors amplification of the enzyme thymidine phosphorylase is about 500 fold; in host cells containing no functional deo R and cyt R repressors, amplification of thymidine phosphorylase is about 50,000 fold.
G. Dandanell and I. K. Hammer, The EMBO Journal 1: 3333-3338 (1985), disclose transcriptional fusion of deo P1 and deo P2 promoter fragments to the Escherichia coli galactokinase gene. Dandanell et al. examined the expression of galactokinase, i.e., a prokaryotic gene product, under the control of the deo P1-P2 promoters integrated into the bacterial chromosome. By contrast, the present invention teaches the use of the deo P1-P2 promoter on multicopy plasmids to drive the expression of eucaryotic gene products.
Dandanell et al. state that the .lambda. P.sub.L promoter is 2-3 times as strong as the deo P2 promoter (p. 3326, column 2). By contrast, the levels of expression obtained by utilization of the specific polypeptides and particular regulatory elements of the subject invention approach or exceed those obtained by using a .lambda. P.sub.L promoter.
By suggesting that the .lambda. P.sub.L promoter is 2 to 3 times stronger than the deo P2 promoter, which in turn is stronger than the deo P1 promoter, Dandanell et al. teach away from the use of deo P1-P2 promoter as a strong promoter. As noted above, applicants have found that when using the plasmids of the subject invention which contain the deo P1-P2 promoter together with the other regulatory components, polypeptides are produced in unexpectedly large amounts.
PCT International Publication No. WO 84 01171, published Mar. 29, 1984, describes, but does not enable, the use of a deo promoter to express very low amounts of the controlling protein of Escherhichia coli replication, i.e., a noneucaryotic protein.
Plasmids expressing gene products under the control of .lambda. P.sub.L promoter, yet independent of .lambda. genes on the host chromosome, have been designated by us as "independent" plasmid expression vectors. M. Mieschendahl, T. Petri and Urs Hanggi, Biotechnology 4: 802-808 (1986), for example, disclose an independent plasmid in which the .lambda. cI repressor gene is controlled by the tryptophan operator/promoter. However, this is not a temperature controlled system.
M. Zabeau and K. K. Stanley, the EMBO Journal 1: 1217-1224 (1982) placed the gene for a temperature-sensitive cI repressor on a low copy plasmid which is compatible with pBR322 vectors. This approach, however, requires that two plasmids be used and that the cI plasmid be kept in the cells by selection.
A. S. Waldman, E. Haeusslein and G. Milman, J. Biol. Chem. 258: 11571-11575 (1983) cloned the gene and authentic promoter for the temperature sensitive cI 857 repressor directly onto a plasmid. However, good expression of cloned genes was not achieved in such constructs due to the amplification of the cI gene and the concomitant increase in the number of cI molecules. In these experiments, the cloned gene product was only about 4% of total, soluble, cellular protein.
Nowhere in the art is there described an efficient temperature-sensitive, expression system where the heat-inducible expression of genes carried on expression vectors is independent of expression of genes on the host chromosome. Thus, there is no disclosure of efficient, temperature-sensitive "independent" plasmid expression vectors.
In one embodiment of the present invention the weak deo P1 promoter obtained from the deo operon is inserted into a .lambda. P.sub.L expression plasmid where it controls the expression of .lambda. cI 857 repressor gene, thus allowing thermoinducible expression of gene products controlled by the .lambda. P.sub.L promoter on the same plasmid. This expression plasmid is thus independent of .lambda. genes on the host chromosome and may be transformed into a wide variety of hosts. It is thus an "independent" expression plasmid. Unexpectedly, expression of desired gene products using this plasmid can be achieved at levels of up to 25% of total cellular protein.